| Acute lung injury(ALI)is a pulmonary edema and inflammation caused by various factors,which is a great threat to human and animal health.At present,most of the drugs for ALI only can delay some symptoms,cannot change the basic process of the disease,and even bring serious side effects.Therefore,it is urgent to find more safe and effective drugs to prevent and treat ALI.Excessive inflammation and oxidative stress are considered to be the key points of the pathogenesis of acute lung injury.Accordingly,targeting inflammatory response and oxidative stress may be the key to prevent and treat acute lung injury.Activation of AMPK/Nrf2 signaling pathway can regulate inflammatory response and oxidative stress effectively.Many natural compounds can activate the AMPK/Nrf2 signaling pathway,which has multiple pharmacological activities and less side effects.In our previous research,we found that rosmarinic acid could effectively alleviate airway inflammation and oxidative lung injury in allergic asthma of mice,but its anti-inflammatory and anti-oxidative mechanisms have not been clarified.Consequently,the aim of this study is to clarify the anti-inflammatory and anti-oxidative effects of RA in lipopolysaccharide(LPS)-induced acute lung injury(ALI)in vitro and in vivo,which will be able to provide a scientific basis for natural compounds in the prevention and treatment of ALI.The present study was designed to investigate the experimental mechanism of rosmarinic acid ameliorating acute lung injury by establishing a mouse model of acute lung injury induced by lipopolysaccharide(LPS).CCK-8 method,molecular docking,immunofluorescence quantitative PCR(RT-q PCR)and Western Blot were used to study the mechanism at the cellular and molecular levels.Besides,the anti-inflammatory and anti-oxidative activities of rosmarinic acid in vitro were deeply revealed,and the interaction between the anti-inflammatory and anti-oxidative was further investigated.To investigate the protective effect of rosmarinic acid,the acute lung injury BALB/c mice model induced by LPS was used to do the further experiment.In addition,on the basis of cell experiment,we also studied the protective mechanism of oxidative stress,inflammatory reaction and downstream-related programmed cell death in ALI mouse model.And the Cross-talk was further explored by specific inhibitors blocking the AMPK/Nrf2 signaling pathway.This study provides a theoretical basis for exploring new targets and possible molecular mechanisms of rosmarinic acid in the prevention and treatment of acute lung injury.In this process,H & E staining,enzyme-linked immunosorbent assay(ELISA),immunofluorescence(IF),TUNEL staining,immunohistochemistry(IHC)and Western blot were used.The main findings of this study are as follows:1.Rosmarinic acid regulates AMPK/Nrf2 and autophagy pathway to exert antioxidant and anti-inflammatory activities.In RAW264.7 cell model stimulated by t-BHP,rosmarinic acid significantly increased the activity of RAW264.7 cells,significantly inhibited the production of reactive oxygen species(ROS),and protected the cells from the attack of oxygen free radicals.Additionally,in RAW264.7 cell model induced by LPS,rosmarinic acid significantly inhibited the production of i NOS,COX2 and the m RNA expression of IL-1β,IL-6 and TNF-α.Rosmarinic acid also significantly alleviated t-BHP-induced oxidative damage and LPS-induced inflammatory response in two models.Furthermore,the present study further revealed that the antioxidant activity of rosmarinic acid was related to the up-regulation of AMPK/Nrf2 and autophagy pathway,and the anti-inflammatory activity of rosmarinic acid was related to the activation of AMPK-m TOR and regulation of autophagy by AMPK/Nrf2 signaling pathway.2.Rosmarinic acid has a good protective effect on acute lung injury in mice.In vivo,rosmarinic acid was shown to reduce lung histopathology and serum levels of inflammatory cytokines in a mouse model of LPS-induced acute lung injury.Besides,the rosmarinic acid inhibited the formation of MDA,MPO and the consumption of SOD,GSH in lung tissue.In addition,rosmarinic acid could effectively inhibit LPS-activated HMGB1-TLR4/MAPK-NF-κB,NLRP3 inflammasome,and the expression of apoptosis-related proteins,thereby effectively inhibiting apoptosis of lung tissue.Furthermore,it can reduce the expression of NOX4 and NOX2,and up-regulate the expression of x-CT and GPX4,thus effectively alleviate the iron death caused by mitochondrial injury in lung tissue.Rosmarinic acid could increase the nuclear translocation of Nrf2,decrease the expression of Keapl and increase the expression of AMPK,GCLC,GCLM,HO-1 and NQO1 in lung tissue.These results showed that,the protective effects of rosmarinic acid may be related to the up-regulation of AMPK/Nrf2 signaling pathway and the inhibition of LPS-induced activation of HMGB1/TLR4,MAPK,NF-κB,NLRP3,apoptosis and ferroptosis pathway.3.Rosmarinic acid attenuates acute lung injury by targeting AMPK/Nrf2 signaling pathway.Compared to ALI mouse model induced by LPS alone,rosmarinic acid significantly attenuated the LPS-induced pathological changes in the lung tissue and inhibited the activation of NF-κB pathway and NLRP3 inflammasome in a murine ALI model in which AMPK/Nrf2 signaling pathway was blocked by the inhibitor.These results suggested that the activation of AMPK/Nrf2 signaling pathway may be a new therapeutic strategy to inhibit lung injury.It was demonstrated that rosmarinic acid could attenuate LPS-induced lung injury by inhibiting inflammation,oxidative stress,autophagy,apoptosis and ferroptosis through AMPK-regulated Nrf2 activation and the expression of downstream antioxidant enzymes.Taken together,rosmarinic acid can activate the AMPK/Nrf2 signaling pathway to alleviate oxidative stress;rosmarinic acid activates autophagy by regulating AMPK and its downstream autophagy-related pathways,thereby exerting an anti-inflammatory effect.In vivo experiments further confirmed that the regulation of rosmarinic acid has multiple mechanisms and multiple targets.NF-κB signaling pathway and inflammasome activation were inhibited by activating AMPK/Nrf2 signaling pathway.And the ferroptosis,apoptosis,and the release of pro-inflammatory factors were inhibited as well.Finally,the acute lung injury in mice was alleviated. |
